The present invention relates to recombinant DNA technology, and in particular to introducing and expressing foreign DNA in a eukaryotic cell.
The Alphavirus genus includes a variety of viruses all of which are members of the Togaviridae family. The alphaviruses include Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86 (S.A.AR86), Girdwood S.A. virus, Ockelbo virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O""Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.
The alphavirus genome is a single-stranded, messenger-sense RNA, modified at the 5xe2x80x2-end with a methylated cap, and at the 3xe2x80x2-end with a variable-length poly (A) tract. The viral genome is divided into two regions: the first encodes the nonstructural or replicase proteins (nsP1-nsP4) and the second encodes the viral structural proteins. Strauss and Strauss, Microbiological Rev. 58, 491-562, 494 (1994). Structural subunits consisting of a single viral protein, C, associate with themselves and with the RNA genome in an icosahedral nucleocapsid. In the virion, the capsid is surrounded by a lipid envelope covered with a regular array of transmembranal protein spikes, each of which consists of a heterodimeric complex of two glycoproteins, E1 and E2. See Paredes et al., Proc. Natl. Acad. Sci. USA 90, 9095-99 (1993); Paredes et al. Virology 187, 324-32 (1993); Pedersen et al., J. Virol. 14:40 (1974).
Sindbis virus, the prototype member of the alphavirus genus of the family Togaviridae, and viruses related to Sindbis are broadly distributed throughout Africa, Europe, Asia, the Indian subcontinent, and Australia, based on serological surveys of humans, domestic animals and wild birds. Kokemot et al., Trans. R. Soc. Trop Med. Hyg. 59, 553-62 (1965); Redaksie, S. Aft. Med. J. 42, 197 (1968); Adekolu-John and Fagbami, Trans. R. Soc. Trop. Med. Hyg. 77, 149-51 (1983); Darwish et al., Trans. R. Soc. Trop. Med. Hyg. 77, 442-45 (1983); Lundstrxc3x6m et al., Epidemiol. Infect. 106, 567-74 (1991); Morrill et al., J. Trop. Med. Hyg. 94, 166-68 (1991). The first isolate of Sindbis virus (strain AR339) was recovered from a pool of Culex sp. mosquitoes collected in Sindbis, Egypt in 1953 (Taylor et al., Am. J. Trop. Med. Hyg. 4, 844-62 (1955)), and is the most extensively studied representative of this group. Other members of the Sindbis group of alphaviruses include South African Arbovirus No. 86, Ockelbo82, and Girdwood S.A. These viruses are not strains of the Sindbis virus; they are related to Sindbis AR339, but they are more closely related to each other based on nucleotide sequence and serological comparisons. Lundstrxc3x6m et al., J. Wildl. Dis. 29, 189-95 (1993); Simpson et al., Virology 222, 464-69 (1996). Ockelbo82, S.A.AR86 and Girdwood S.A. are all associated with human disease, whereas Sindbis is not. The clinical symptoms of human infection with Ockelbo82, S.A.AR86, or Girdwood S.A. are a febrile illness, general malaise, macropapular rash, and joint pain that occasionally progresses to a polyarthralgia sometimes lasting from a few months to a few years.
The study of these viruses has led to the development of beneficial techniques for vaccinating against the alphavirus diseases, and other diseases through the use of alphavirus vectors for the introduction of foreign DNA. See U.S. Pat. No. 5,185,440 to Davis et al., and PCT Publication WO 92/10578. It is intended that all United States patent references be incorporated in their entirety by reference.
It is well known that live, attenuated viral vaccines are among the most successful means of controlling viral disease. However, for some virus pathogens, immunization with a live virus strain may be either impractical or unsafe. One alternative strategy is the insertion of sequences encoding immunizing antigens of such agents into a vaccine strain of another virus. One such system utilizing a live VEE vector is described in U.S. Pat. No. 5,505,947 to Johnston et al.
Sindbis virus vaccines have been employed as viral carriers in virus constructs which express genes encoding immunizing antigens for other viruses. See U.S. Pat. No. 5,217,879 to Huang et al. Huang et al. describes Sindbis infectious viral vectors. However, the reference does not describe the cDNA sequence of Girdwood S.A. and TR339, nor clones or viral vectors produced therefrom.
Another such system is described by Hahn et al., Proc. Natl. Acad. Sci. USA 89:2679 (1992), wherein Sindbis virus constructs which express a truncated form of the influenza hemagglutinin protein are described. The constructs are used to study antigen processing and presentation in vitro and in mice. Although no infectious challenge dose is tested, it is also suggested that such constructs might be used to produce protective B- and T-cell mediated immunity.
London et al., Proc. Natl. Acad; Sci, USA 89, 207-11 (1992), disclose a method of producing an immune response in mice against a lethal Rift Valley Fever (RVF) virus by infecting the mice with an infectious Sindbis virus containing an RVF epitope. London does not disclose using Girdwood S.A. or TR339 to induce an immune response in animals.
Viral carriers can also be used to introduce and express foreign DNA in eukaryotic cells. One goal of such techniques is to employ vectors that target expression to particular cells and/or tissues. A current approach has been to remove target cells from the body, culture them ex vivo, infect them with an expression vector, and then reintroduce them into the patient.
PCT Publication No. WO 92/10578 to Garoff and Liljestrom provide a system for introducing and expressing foreign proteins in animal cells using alphaviruses. This reference discloses the use of Semliki Forest virus to introduce and express foreign proteins in animal cells. The use of Girdwood S.A. or TR339 is not discussed. Furthermore, this reference does not provide a method of targeting and introducing foreign DNA into specific cell or tissue types.
Accordingly, there remains a need in the art for full-length cDNA clones of positive-strand RNA viruses, such as Girdwood S.A and TR339. In addition, there is an ongoing need in the art for improved vaccination strategies. Finally, there remains a need in the art for improved methods and nucleic acid sequences for delivering foreign DNA to target cells.
A first aspect of the present invention is a method of introducing and expressing heterologous RNA in bone marrow cells, comprising: (a) providing a recombinant alphavirus, the alphavirus containing a heterologous RNA segment, the heterologous RNA segment comprising a promoter operable in bone marrow cells operatively associated with a heterologous RNA to be expressed in bone marrow cells; and then (b) contacting the recombinant alphavirus to the bone marrow cells so that the heterologous RNA segment is introduced and expressed therein.
As a second aspect, the present invention provides a helper cell for expressing an infectious, propagation defective, Girdwood S.A. virus particle, comprising, in a Girdwood S.A.-permissive cell: (a) a first helper RNA encoding (i) at least one Girdwood S.A. structural protein, and (ii) not encoding at least one other Girdwood S.A. structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding the at least one Girdwood S.A. structural protein encoded by the first helper RNA, and (ii) encoding the at least one other Girdwood S.A. structural protein not encoded by the first helper RNA, and with all of the Girdwood S.A. structural proteins encoded by the first and second helper RNAs assembling together into Girdwood S.A. particles in the cell containing the replicon RNA; and wherein the Girdwood S.A. packaging segment is deleted from at least the first helper RNA.
A third aspect of the present invention is a method of making infectious, propagation defective, Girdwood S.A. virus particles, comprising: transfecting a Girdwood S.A.-permissive cell with a propagation defective replicon RNA, the replicon RNA including the Girdwood S.A. packaging segment and an inserted heterologous RNA; producing the Girdwood S.A. virus particles in the transfected cell; and then collecting the Girdwood S.A. virus particles from the cell. Also disclosed are infectious Girdwood S.A. RNAs, cDNAs encoding the same, infectious Girdwood S.A. virus particles, and pharmaceutical formulations thereof.
As a fourth aspect, the present invention provides a helper cell for expressing an infectious, propagation defective, TR339 virus particle, comprising, in a TR339-permissive cell: (a) a first helper RNA encoding (i) at least one TR339 structural protein, and (ii) not encoding at least one other TR339 structural protein; and (b) a second helper RNA separate from the first helper RNA, the second helper RNA (i) not encoding the at least one TR339 structural protein encoded by the first helper RNA, and (ii) encoding the at least one other TR339 structural protein not encoded by the first helper RNA, and with all of the TR339 structural proteins encoded by the first and second helper RNAs assembling together into TR339 particles in the cell containing the replicon RNA; and wherein the TR339 packaging segment is deleted from at least the first helper RNA.
A fifth aspect of the present invention is a method of making infectious, propagation defective, TR339 virus particles, comprising: transfecting a TR339-permissive cell with a propagation defective replicon RNA, the replicon RNA including the TR339 packaging segment and an inserted heterologous RNA; producing the TR339 virus particles in the transfected cell; and then collecting the TR339 virus particles from the cell. Also disclosed are infectious TR339 RNAs, cDNAs encoding the same, infectious TR339 virus particles, and pharmaceutical formulations thereof.
As a sixth aspect, the present invention provides a recombinant DNA comprising a cDNA coding for an infectious Girdwood S.A. virus RNA transcript, and a heterologous promoter positioned upstream from the cDNA and operatively associated therewith. The present invention also provides infectious RNA transcripts encoded by the above-mentioned cDNA and infectious viral particles containing the infectious RNA transcripts.
As a seventh aspect, the present invention provides a recombinant DNA comprising a cDNA coding for a Sindbis strain TR339 RNA transcript, and a heterologous promoter positioned upstream from the cDNA and operatively associated therewith. The present invention also provides infectious RNA transcripts encoded by the above-mentioned cDNA and infectious viral particles containing the infectious RNA transcripts.
The foregoing and other aspects of the present invention are described in the detailed description set forth below.